i) Field of the Invention
This invention relates to a monoclonal antibody specific to a substance derived and purified from a human placenta and having anticoagulant activity (hereinafter abbreviated as "PCI"), a hybridoma secreting the monoclonal antibody, and utilization of the monoclonal antibody.
ii) Description of the Prior Art
The cell fusion technology has been developed rapidly since the report of Kohler and Milstein [Nature, 495-497 (1975)]. It has been known that a hybridoma obtained by fusing mammalian spleen cells and myeloma cells secretes various antibodies depending on the characteristics of the spleen cells employed. It has also been attempted to form a hybridoma, which secretes a monoclonal antibody against various biological substances such as proteins and hormones, by effecting cloning based on the characteristics of the hybridoma and also to secrete the monoclonal antibody [E. Dale Servier et al., Clinical Chemistry, 27(11), 1797-1806 (1981)].
In the meantime, the present applicant previously succeeded in isolating and purifying a substance having anticoagulant activity (PCI) from a human placenta and applied for a patent thereon (European Patent Application Publication No. 0217341). PCI is a substance which is substantially free from glucose, has the following properties and is useful as a medicine.
(1) Molecular weight (SDS-polyacrylamide gel electrophoresis, reduced state): 34,000.+-.2,000. PA1 (2) Isoelectric point (isoelectric column electrophoresis using an ampholyte): 4.7.+-.0.1. PA1 (4) Effects: PA1 (5) Analysis of amino acids in mole percent:
(a) Inactivated by a heat treatment at 50.degree. C. for 30 minutes. PA2 (b) Stable in a pH range of 4-10. PA2 (c) Stable in plasma at 37.degree. C. for 30 minutes. PA2 (a) Capable of prolonging the recalcification time. PA2 (b) Capable of prolonging the prothrombin time. PA2 (c) Capable of prolonging the activated partial thromboplastin time. PA2 The existence of aspartic acid 9.8, threonine 7.0, serine 6.3, glutamic acid 13.3, proline 2.0, glycine, 7.5, alanine 8.0, 1/2 cystine 0.3, valine 4.6, methionine 1.8, isoleucine 5.3, leucine 11.8, tyrosine 3.6, phenyl-alanine 4.2, histidine 1.4, lysine 7.2 and arginine 6.2 is recognized by the analysis of amino acids.
One example of preparation of PCI will be described subsequently in Example 1. It may be summarized as follows.
A placenta homogenate is first prepared from the human placenta and then subjected to centrifugation. The homogenization is effected in the following manner. After cutting off the amnion and the like from the placenta, the placenta is washed thoroughly with a physiological saline, followed by homogenization by the use of a Waring blender and "Polytron" (trade mark; manufactured by Kinema SA). The thus-obtained homogenate was subjected to centrifugation, thereby obtaining a supernatant and sediment. The resulting placenta homogenate sediment is washed thoroughly with a buffer and is then subjected again to centrifugation to obtain a washed sediment, followed by extraction. Namely, the thus-obtained sediment of the placenta homogenate is immersed in a buffer, which contains a chelating agent such as EDTA, EGTA, oxalic acid, citric acid, sodium nitrilotriacetate or phosphoric acid, and/or another buffer containing a surfactant such as "Triton X-100", Lubrol (trade mark), SDS, deoxycholic acid or the like. After allowing it to stand overnight at 4.degree. C.-8.degree. C., the mixture is centrifuged to collect a supernatant as an extract. Here, the extraction may be carried out by using both chelating agent and a surfactant.
The supernatant is subjected further to ultracentrifugation at 50,000 to 100,000 .times. g to obtain a microsome fraction as a sediment. After washing the microsome fraction, it was extracted with a chelating agent and/or a surfactant in the same manner as described above and the resultant extract was subjected to ultracentrifugation to collect a supernatant as an extract.
The thus-obtained extract is subjected to ammonium sulfate fractionation. The ammonium sulfate fractionation is effected in the following manner. First, solid ammonium sulfate is added to 35% of its saturated concentration to the extract, followed by centrifugation to collect a supernatant. Ammonium sulfate is then added to the supernatant until its concentration reached 85% of its saturated concentration, followed by centrifugation to collect a sediment.
The resulting ammonium sulfate fraction is then purified by known isolation and purification procedures including, for example, dialysis, ion exchange chromatography, gel filtration, adsorption chromatography, hydrophobic chromatography, isoelectric point column electrophoresis, affinity chromatography using lectin or an antibody, and the like either singly or in combination, thereby obtaining PCI. For example, a fraction obtained by subjecting the chelating agent and/or surfactant extract to ammonium sulfate fractionation is dialyzed thoroughly. The resulting dialyzate is then eluted in accordance with the linear concentration gradient method in which "DEAE-Toyopearl" (trade name) is used. After an active fraction thus obtained is dialyzed, it is caused to pass through "Blue Sepharose" (trade name; product of Pharmacia AB). The active fraction is then concentrated and subjected to gel filtration through "Sephadex G-100" (trade name; product of Pharmacia AB), thereby to obtain PCI.
PCI is however contained only in a trace amount in the placenta, and no sufficient specific binding is established with PCI in various chromatographic techniques which are employed routinely. It is hence difficult to obtain PCI in a highly pure form. Moreover, such conventional procedures require many steps and are unable to achieve any satisfactory recovery rate.
It has therefore been desired to develop a specific purification process for obtaining high-purity PCI easily at a high recovery rate. In order to use PCI as an anticoagulant, it has also been desired to elucidate the mechanism of the action of PCI and further to develop a high-sensitivity assay for PCI as a method for measuring its blood level.